Novel microbial acidic heteropolysaccharide and production thereof

ABSTRACT

METHOD FOR PRODUCING MICROBIAL ACIDIC HETEROPOLYSACCHARIDE, OF WHICH THE SACCHARIDE UNITS CONSIST SUBSTANTIALLY OF GLUCOSE AND MANNOSE AND THE ACID UNITS CONSIST SUBSTANTIALY OF SUCCINIC ACID, LACTIC ACID AND ACETIC ACID, WHICH COMPRISES INCUBATING CORYNEBACTERIUM EQUI VAR. MUCILAGINOSUS IN A CULTURE MEDIUM CONTAINING HYDROCARBON AS THE MAIN CARBON SOURCE TOGETHER WITH DIGESTIBLE NITROGEN SOURCE AND OTHER NUTRIENTS NECESSARY FOR THE GROWTH OF THE MICROORGANISM UNTIL A SUBSTANTIAL AMOUNT OF THE ACIDIC HETEROPOLYSACCHARIDE IS ACCUMULATED IN THE CULTURE BROTH, AND RECOVERING THE ACCUMULATED ACIDIC HETEROPOLYSACCHARIDE THEREFROM.

July 4, 1972 sABuRo YAMAToDANl ETAL 3,674,642

NOVEL MICROBIAL ACIDIC HETEROPOLYSACCHARIDE AND PRODUCTION THEREOF Filed May 19, 1970 (maoaad) aamvmwsNvai INVENTORS SABURO YAMATODANI TSUNEO KANAMURU ATTORNEYS United States Patent 3,674,642 NOVEL MICRBIAL ACIDIC HETEROPOLY- SACCHARIDE AND PRODUCTION THEREOF Saburo Yamatodani, Minoo, and Tsuneo Kanamaru,

Toyonaka, Iapan, assignors to Takeda Chemical Industries, Ltd., Osaka, Japan Filed May 19, 1970, Ser. No. 38,780 Claims priority, application Japan, May 19, 1969, 44/ 38,551 Int. Cl. C1211 13/04 U.S. Cl. 195-28 R 4 Claims ABSTRACT F THE DISCLOSURE Method for producing microbial acidic heteropolysaccharide, of which the saccharide units consist substantially of glucose and mannose and the acid units consist substantialy of succinic acid, lactic acid and acetic acid, which comprises incubating Cozynebacterium equi var. muclaginosus in a culture medium containing hydrocarbon as the main carbon source together with digestible nitrogen source and other nutrients necessary for the growth of the microorganism until a substantial amount of the acidic heteropolysaccharide is accumulated in the culture broth, and recovering the accumulated acidic heteropolysaccharide therefrom.

The present invention relates to a novel microbial acidic heteropolysaccharide and a, method for the production of the same.

This invention is based on the following iindings:

(l) Corynebacterium equi. var. mucilagnosus, a nefw Variety of Corynebacterium equi isolated from a sample of soil, accumulates a novel acidic heteropolysaccharide when the microorganism is incubated in a culture medium containing a hydrocarbon as the main carbon source;

(2) The so-accumulated acidic heteropolysaccharide can be recovered in a desired purity from the culture broth, utilizing its physicochemical properties; and

(3) The acidic heteropolysaccharide shows a strong vis-cosity and is excellent as an adhesive, for instance.

Thus, the principal object of the present invention is to provide novel acidic heteropolysaccharide which may -be used, e.g. as an excellent adhesive.

Another object of the present invention is to provide an industrially feasible method for producing said acidic heteropolysaccharide.

The acidic heteropolysaccharide of the present invention is characterized by the following properties:

The saccharide units of the acidic heteropolysaccharide consist substantially of glucose and mannose, and the acid units thereof consist substantially of succinic acid, lactic acid and acetic acid.

The specic rotation of the lacidic heteropolysaccharide is [a]D25=l-35i5 (c.\=0.5%, in water).

The acidic heteropolysaccharide shows a high viscosity. For instance, the viscosity of its 0.5% aqueous solution as measured employing Ubbelohdes viscometer is higher than about 5,000 centipoises. The intrinsic viscosity of the acidic heteropolysaccharide falls within the range of about 25 to about 28.

The acidic heteropolysaccharide shows significant infrared absorption bands in KBr disc method at the wave length of 12.8, 11.6, 6.23 and 5.75 microns.

The Weight ratio of the saccharide units in terms of their free form; the total of succinic acid and lactic acid in terms of their free forms; and acetic acid calculated as OHgCOO-group; is about 70-85; 8-12; 8-15. The saccharid'e units contain about 50% to about 75% of glucose 3,674,642 Patented July 4, 1972 ice (l) Morphological. characteristics (l) Vegetative cells:

Shape: Cocci to elongated rods. Depending upon type of medium and duration of incubation, rnost of the cells occur in irregular chains or branching, occasionally single or pair.

Motility: Nonmotile.

Size: 1.0 to 1.3 microns by 1.0 to 3.0 microns.

Sporulation: Nonsporulating.

(2) Grams stain: Positive and unchanged even after a long incubation period.

(Il) Cultural characteristics (l) Bouillon (24 to 48 hours at 28 C.): Light reddish brown pellicles are formed, with fair amounts of light reddish sediment. Medium growth.

(2) Bouillon slant (48 hours at 28 C.): Growth abundant filamentous and raised; glistening and smooth. Light reddish pink to light reddish brown. No diffusible pigment.

(3) Bouillon stab: Growth abundant on surface; papillose and uprising.

(4) Nutrient agar plate (48 hours at 28 C.): Entire circular (1.4 to 2.2 millimeters in diameter), convex to capitate. Smooth and glistening surface, reddish brown with a hint of light yellow to light reddish pink.

(5) Gelatin stab (30 days at 28 C.): Surface growth.

No liquefaction.

(III) Physiological characteristics (l) Temperature relations: Growth at 20 to 45 C.,

optimum about 37 C.

(2) pH relations: Growth at pH 5.0 to 9.0, optimum about 6.5 to 8.5

(3) O2 relations: Aerobic (4) Indol: Not produced (5) Nitrate reduction: lNitrites produced from nitrates (6) Hydrogen sulde: Produced (7) `Starch assimilation: Negative (8) Methyl red test: Negative (9) Voges-Proskauer reaction: Negative (10) Catalase: Produced (ll) Production of ammonia from peptone: Very slight (l2) Litmus milk: No change (13) Ammonium salts, nitrates and urea are utilized as nitrogen sources (14) Utilization of carbon sources:

Growth on glucose, succinic acid, glycerol, propionic acid, pyruvic acid, acetic acid, benzoic acid and normal paraiiins from 8 to 19 carbon atoms asl the sole carbon source. Gluconic acid, citric acid, 2-ketogluconic acid and malonic acid are not utilized as the sole carbon source. No acid and no gas are produced from glucose, fructose, galactose, mannose, Xylose, arabinose, sucrose, lactose, maltose, trehalose, ranose, mannitol, glycerol, dextrin, starch, inulin and glycogen.

Detailed comparison of the above-mentioned characteristics with the description in Bergeys Manual of Determinative Bacteriology, seventh edition reveals that this microorganism belongs to the genus Corynebacterium and that these characteristics are substantially the same as those of Corynebacterium equi. However, though the type culture of Corynebacterium equi (IFO No. 3730) gives good growth in a culture medium containing normal parafiins as the sole carbon source, the resultant culture broth is substantially not viscous, nor does it show signs of any substantial accumulation of mucillagenous material. In sharp contrast thereto, this microorganism accumulates the above-mentioned acidic heteropolysaccharide to give a culture broth of a high viscosity when incubated in a culture medium containing a hydrocarbon as the main carbon source. Therefore, this microorganism has been recognized as a variety of Corynebacterium equi, and named Corynebacterium equi var. muclagnosus.

According to the method of the present invention, Corynebacterium equi var. muclaginosus is incubated in a culture medium containing hydrocarbon, particularly normal parafiins, as the main carbon source. Especially advantageous hydrocarbons are normal parafiins of carbon atoms of 13 to 19 and their typical examples include tetradecane, hexadecane, octadecane, nonadecane, and n-paraffin mixture which boils in the range of about 230 to about 330 C. and the like.

-From the viewpoints of the growth of the microorganism and the yield of the objective acidic heteropolysaccharide, there may be advantageously employed a culture medium containing about 2 to about 15 percent by volume of normal paraflins having carbon atoms of 13 to 19.

As these hydrocarbons are scarcely soluble in water, the addition thereof to an aqueous culture medium is practically carried out under shaking to prepare a suspension. If desired, a suspending agent, e.g. a surfactant, may be employed.

It is sufficient for the present method to use these hydrocarbons as carbon sources, but, if desired, any other conventional carbon sources such as carbohydrates (eg. glucose, mannose), acetic acid, glycerol, succinic acid and the like may be employed.

The culture medium should contain nitrogen sources(s) together with the hydrocarbon as nutrients. The nitrogen sources may be any of conventional ones such as peptone, soybean powder, corn steep liquor, meat extract, yeast extract, ammonium salts, nitrates, urea and the like.

Furthermore, there are added to the medium inorganic salts which are commonly used in the incubation of microorganisms, such as the phosphate of potassium or sodium, as well as the sulfates, hydrochloride and/or carbonates of metals, e.g. magnesium, iron, manganese, calcium, zinc, cobalt, etc. Aside from the foregoing, it is of course possible to add as required, such trace growth factors as vitamins and nucleic acid related compounds.

The culture medium may be used in any form, but a liquid medium is generally advantageous. When a liquid medium is employed, any of shaking cultural method, stirring cultural method or the like may be adopted, though the so-called submerged aerobic cultural method is the most desirable for the industrial process.

Incubation conditions such as the pH of the medium and the incubation temperature should be controlled so as to produce the objective acidic heteropolysaccharide in the maximum amount. Advantageously, the pH of the culture medium and the incubation temperature are respectively adjusted to pH about 5 to about `8, especially to pH about 6-7, and about 25 to about 40 C. As the pH of the medium changes to acidic region with the progress of incubation, the pH of the medium is practically maintained through the incubation period within said range with the previous incorporation of calcium carbonate into the medium or by adding an alkali hydroxide solution or aqueous ammonia to the medium.

Although the period required for the maximum accumulation of the acidic heteropolysaccharide is changeable depending upon various factors, the substantial amount of the heteropolysaccharide accumulates in the culture broth usually between about 48 to about 120 hours from the start of the incubation.

Generally-known means for recovering acidic heteropolysaccharides from the culture broth containing them can be applied to the recovery of the acidic heteropolysaccharide of the present invention from the culture broth. The acidic heteropolysaccharide can be absorbed on various adsorbents, or precipitated with some precipitants. Moreover, conventional means for recovery, such as salting out or dialysis or a combination thereof, may be employed for the purpose of recovery and purification.

Most practically, the viscous culture broth is diluted fivefold or more with water to lessen its viscosity and subjected to centrifugation or filtration by employing a filter press with a filter aid to remove the microorganism cells and other solid materials. In this step, by warming the culture broth at about 50 to about 95 C., the amount of water as the diluent may be reduced and filtration facilitated.

Thus-obtained clear solution is concentrated as much as possible. To the concentrate is added hydrophilic organic solvent such as acetone, methyl alcohol, ethyl alcohol up to a concentration of about 60 to about 80% by volume, or alternatively, a small amount of sodium chloride is added first and a hydrophilic organic solvent is subsequently added up to a concentration of about 50 to about 60% by volume, whereby the objective acidic heteropolysaccharide precipitates in the form of a white spongy sediment. Salting out with a suitable organic solvent such as ammonium sulfate, sodium sulfate may also be employed for the separation of the acidic heteropolysaccharide from the above-mentioned concentrate.

Alternatively, the broth filtrate after removal of the microorganism cells may be concentrated to a certain extent and subjected to spray drying or drum drying to give a crude preparation of the acidic heteropolysaccharide.

Though thus-obtained crude acidic heteropolysaccharide may be employed as it is, the crude heteropolysaccharide may further be purified. The purification may be carried out by, for example, further subjecting the crude acidic heteropolysaccharide to the above-mentioned precipitation treatments with the hydrophilic organic solvent, and then to dialysis or to treatments with strongly acidic cation exchange resins, and finally to lyophilization.

The acidic heteropolysaccharide of the present invention has many superior properties, particularly an increased viscosity, as compared with hitherto-known polysaccharides. For instance, the viscosity of its 0.5% aqueous solution as measured employing Ubbelohdes viscometer at 30 C. is higher than about 5,000 centipoises and occasionally reaches about 20,000 centipoises.

The acidic heteropolysaccharide of the present invention may be employed, for example, as an adhesive. When employed as an adhesive, about 2 to about 5% aqueous solution is preferable. The acidic heteropolysaccharide is very excellent as an adhesive because it can exhibit a strong adhesion in a low concentration and it is not hygroscopic. For instance, the acidic heteropolysaccharide shows a strong adhesion in one-fourth concentration of gum arabic as shown in test described below.

The following examples and test are merely for illustrative purposes and are not to be construed as the limitation of the present invention. Throughout the present specification, the abbreviations ml., 1., mcg., g.," kg and C. mean milliliter(s), liter(s), microgram(s), gram(s), kilogram(s) and centigrade(s), respectively, and percent is weight per volume excepting the case Where the meaning is clearly otherwise from the context. The ATCC No. indicates the accession number of the microorganism deposited at the American Type Culture Collection in Rockville, Md., U.S.A.

EXAMPLE l Corynebacterium equi var. mucilagnosus (ATCC No. 21521) is inoculated into 30 ml. of an aqueous medium comprising 1% of glucose, 0.5% of yeast extract, 0.5% of peptone, 0.5% of NaCl (pH 7.0) and is incubated under shaking at 28 C. for 24 hours.

l ml. of the resulting culture broth is inoculated into 100 ml. of the culture medium mentioned in the following Table 1, and is incubated under shaking at 28 C. for 96 hours.

The resulting culture broth of 7.4 of relative viscosity is diluted tenfold with Water and the diluted broth is subjected to centrifugation at 10,000 r.p.m. for 2O minutes to remove the microorganism cells and the residual calcium carbonate. The supernatant is concentrated to onetwentieth of the original volume. To the concentrate is added acetone in 3 to 4 times the volume of the concentrate with stirring, whereby the heteropolysaccharide separates as occulent precipitates. The precipitates are recovered by filtration. The precipitates are dehydrated with 150 ml. of acetone and then dried to give white spongy crude heteropolysaccharide. The yield is 1.55 g. or 20.1% based on the weight of the n-parain mixture used.

The crude heteropolysaccharide is dissolved in 1 litre of water and the solution is subjected to dialysis with deionized 'water at 20 C. for 48 hou-rs. Thus-treated solution is allowed to pass through a column (5 cm. x 50 cm.) packed with strongly acidic cation exchange resin (H- form, such as Amberlite IRO-120) and the eluent is lyophilized to give white spongy heteropolysaccharide. The yield is 1.06 g. or 13.8% based on the weight of the nparaiin mixture used.

Thus-obtained preparation of the acidic heteropolysaccharide has the following properties:

(l) Total Weight of glucose units and mannose units is 81.09% by weight relative to the whole weight of the heteropolysaccharide and weight ratio of glucose units and mannose units is 3:2 in terms of their free form, when measured by hydrolyzing the heteropolysaccharide preparation with 2 N sulfuric acid at 55 C. for 5 hours and subjecting the hydrolyzate to paper chromatography (developer A: a mixture of n-butyl alcohol, pyridine and Water in 6:4:3, developer B: a mixture of n-butyl alcohol, acetic acid and water in 3:l:1, ascending method) and to gas chromatography Iwith N2 gas employing authentic D- glucose and D-mannose as control.

Total weight of succinic acid and lactic acid is 8.99% relative to the Whole weight of the heteropolysaccharide and the weight ratio of lactic acid relative to succinic acid is about 0.18 in terms of their free form, when measured by methylatiug the diethyl ether-soluble fraction of the above-mentioned hydrolyzate with diazomethane and subjecting the methylated fraction to gas chromatography with N2 gas employing cyclohexanol as control.

Acetic acid is '9.99% calculated as CHaiCOO-group relative to the whole Weight of the heteropolysaccharide, when measured by the method described in Methods in Carbohydrate Chemistry vol. V, pp. 187 and 188, published by Academic Press, New York, in 1965.

(2) Its specic rotation is [a]D25=-{35 (C=0.5%, in Water).

(3) The viscosity of its 0.5% aqueous solution as measured employing Ubbelohdes Viscometer is 17,500 centipoises.

(4) Its intrinsic viscosity is about 26.

(5) Its infrared absorption spectrum measured by KBr disc method is as shown in the figure of the accompanying drawing.

(6) It reacts with quaternary ammonium salts such as cetylpyridiniurn chloride and cetyltrimethylammonium bromide to make white complex according to the method described in the said Methods in Carbohydrate Chemistry vol. V, pp. 38 to 44, this clearly means that the heteropolysaccharide of the present invention is acidic one.

EXAMPLE 2 Corynebacterium equi var. muclagz'nosus (ATCC 21521) is inoculated into 30 ml. of an aqueous medium comprising 1% of glucose, 0.5 of yeast extract, 0.5 of peptone and 0.5% of NaCl (pH 7.0) and is incubated under shaking at 28 C. for 24 hours.

10 ml. of the resulting culture broth is inoculated into m1. of the culture medium mentioned in the followiny Table 2, and is incubated under shaking at 28 C. for

The resulting culture broth of a high viscosity is subjected to the same isolation procedures as described in Example l to give white spongy crude heteropolysaccharide. The yield is 1.72 g. or 22.2% based on the Weight of the n-parain mixture used.

The crude heteropolysaccharide is further subjected to the same purification treatments as described in Example 1 to give purified heteropolysaccharide. The yield is 0.98 g. or 12.6% based on the weight of the n-parain mixture used.

EXAMPLE 3 Colynebacterium equi var. mucilgnosus (ATCC 21521) is pre-cultured in a 50 litres tank containing 30 litres of an aqueous medium comprising 1% of glucose, 0.5% of yeast extract, 0.5% of peptone and 0.5% of NaCl (pH 7.0) for 24 hours under the conditions of 28 C., 100% aeration and agitation at 250 r.p.m.

l0 litres of the resulting culture broth is inoculated in a 200 litres tank containing 100 litres of a culture medium of the composition listed in the following Table 3 and incubated for 96 hours under the conditions of 30 C., 50% aeration and agitation at 250 r.p.m., during which time the pH of the medium is maintained at about 6.5 with the addition of 30% sodium hydroxide.

TABLE 3 Percent n-Parafn mixture3 (v./v.) 7 NH4NO3 0.2 K2HPO.,l 0.5 KH2PO4 0.2 MgSO4-7H2O 0.1 Peso.; MnSO4'7H2O 0.002 ZnClZ 0.001 COClZ 0.001 Corn steep liquor 0.03 CaCO3 0.3 pH 7.0.

3The n-parain mixture has the same composition as that of the n-paran mixture described in Table 1.

90 litres of the resulting culture broth of a high viscosity is diluted fourfold with water. The diluted broth is heated at 90 C. under stirring for 10 minutes and is quickly ltered through a lter press along with 3. kg. of a filter aid (Hyflo Super Cel produced by Johns-Manville Products Corp. U.S.A.), whereby a clear viscous fluid is obtained. The fluid is concentrated to about 70 litres and |1% by volume of the uid of sodium chloride is added thereto, followed by the addition of 2 to 3 times of acetone, whereby the heteropolysaccharide separates as white spongy precipitates. The spongy precipitates recovered by filtration are dehydrated with 30 litres of acetone and dried to give white spongy crude heteropolysaccharide. The yield is 1.58 kg. or 29.4% based on the weight of the n-parafiin mixture used.

TEST

0.5 ml. of the respective aqueous solutions of the acidic heteropolysaccharide obtained in Example 1 and gum arabic described in Japanese Pharmacopoeia VII in concentrations listed in Table 4 were daubed on each sheet of white paper (5 cm. x 7 cm.) and each sheet was pasted upon a fresh paper sheet of the same shape. After being kept at C. for 24 hours, it was tested whether or not thus-pasted paper sheets could be separated from each other. The results are summarized in Table 4.

Norm-Substantially no adhesion, or pasted paper sheets could bc separated from each other. -i- =Pasted paper sheets couldnot be separated from nach other. (N)=Samp1cs were unable to be obtalned due to very high viscosity.

Table 4 clearly demonstrates that the acidic heteropolysaccharide of the present invention exhibits a. strong adhesion in one-fourth concentration of gum arabic.

We claim:

1. A method for producing microbial acidic heteropolysaccharide, of which the saccharide units consist substantially of glucose and mannose and the acid units consist substantially of succinic acid, lactic acid and acetic acid, which comprises incubating Carynebacterum equi var. mucz'laginosus ATCC No. 21521 in a culture medium containing hydrocarbon as the main carbon source together with digestible nitrogen source and other nutrients necessary for the growth of the microorganism until a substantial amount of the acidic hcteropolysaccharide is accumulated in the culture broth, and recovering the accumulated acidic heteropolysaccharide therefrom.

2. A method according to claim 1, wherein the incubation is carried out at a temperature of about 25 to about 40 C. and at a pH of about 5 to about 8 under aerobic conditions.

3. A method according to claim 1, wherein the culture medium contains about 2 to about 15 percent by volume of normal paraflins.

4,. A method according to claim 3, wherein the normal parafiins have carbon atoms ranging from 13 to 19.

References Cited UNITED STATES PATENTS 3,406,114 10/1958 yGoren 195-31 P A. LOUIS MONACELL, Primary Examiner G. M. NATH, Assistant Examiner 

